Antisense oligonucleotides targeted against human immunodeficiency virus

ABSTRACT

The present invention is directed to oligonuclegtides comprising nucleotide sequences sufficiently complementary to conserved regions of human immunodeficiency virus genetic material such that when bound to said region, the oligonucleotides effectively prevent expression of the genetic material.

This application is a continuation of Ser. No. 08/782,982, filed Jan.14, 1997, now U.S. Pat. No. 5,811,537, which is a continuation of Ser.No. 08/308,869, filed Sep. 19, 1994, now U.S. Pat. No. 5,594,122, whichis a continuation of Ser. No. 08/081,572, filed Jun. 23, 1993, nowabandoned.

FIELD OF THE INVENTION

This invention relates to oligonucleotide (ODN) based therapeutics,particularly the treatment of infections of the human immunodeficiencyvirus (HIV).

BACKGROUND OF THE INVENTION

The present invention relates to ODNs suitable for use in treatment ofHIV infected individuals by inhibition of replication of HIV in infectedcells.

HIV is responsible for the disease that has come to be known as acquiredimmune deficiency syndrome (AIDS). Although initially recognized in1981, no cure has yet been found for this inevitably fatal disease. HIVis spread by a variety of means such as sexual contact, infected bloodor blood products and perinatally. Because of the complexity of HIVinfection and the paucity of effective therapies, a great deal of efforthas been expended in developing methods for detecting, treating andpreventing infection. Diagnostic procedures have been developed foridentifying infected persons, blood and other biological products.

The HIV genome has been well characterized. Its approximately 10 kbencode sequences containing regulatory segments for HIV replication aswell as the gag, pol and env genes coding for the core proteins, thereverse transcriptase-protease-endonuclease, and the internal andexternal envelope glycoproteins, respectively. HIV tends to mutate at ahigh rate causing great genetic variation between strains of the virusesand indeed between virus particles of a single infected individual.There are a few “conserved” regions of the HIV genome which tend not tomutate. These regions are presumed to encode portions of proteinsessential for virus function which can thus withstand very fewmutational events.

The HIV env gene encodes the glycoprotein, gpl60, which is normallyprocessed by proteolytic cleavage to form gp120, the external viralglycoprotein, and gp41, the viral transmembrane glycoprotein. Thegpl120remains associated with HIV virions by virtue of noncovalentinteractions with gp41. These noncovalent interactions are weak,consequently most of the gpl120 is released from cells and virions in asoluble form.

Like most viruses, HIV often elicits the production of neutralizingantibodies. Unlike many other viruses and other infectious agents forwhich infection leads to protective immunity, however, HIV specificantibodies are insufficient to halt the progression of the disease.Therefore, in the case of HIV, a vaccine that elicits the immunity ofnatural infection could prove to be ineffective. In fact, vaccinesprepared from the HIV protein gp160 appear to provide little immunity toHIV infection although they elicit neutralizing antibodies. The failureto produce an effective anti-HIV vaccine has led to the prediction thatan effective vaccine will not be available until the end of the 1990's.Therapeutic agents currently used in treatment of AIDS often causesevere side-effects which preclude their use in many patients. It would,thus, be useful to have alternative methods of treating and preventingthe disease that do not entail vaccination and currently availablepharmaceutical agents.

Recently, attempts have been made to moderate protein productionassociated with viral infections by interfering with the mRNA moleculesthat direct their synthesis. By interfering with the production ofproteins, it has been hoped to effect therapeutic results with maximumeffect and minimal side effects. It is the general object of such atherapeutic approach to interfere with or otherwise modulate geneexpression leading to undesired protein formation.

One method for inhibiting specific gene expression which is believed tohave promise is the “antisense” approach. Single-stranded nucleic acid,primarily RNA, is the target molecule for ODNs that are used to inhibitgene expression by an antisense mechanism. A number of workers havereported such attempts: Stein and Cohen (1988) Cancer Res.,48:2659-2668; Walder (1988) Genes & Development, 2:502-504;Marcus-Sekura (1988) Anal. Biochem., 172:289-295; Zon (1987) J. Pro.Chem., 6:131-145; Zon (1988) Pharm. Res., 5:539-549; Van der Krol et al.(1988) Biotechniques, 6:958-973; and Loose-Mitchell (1988) TIPS,9:45-47. Antisense ODNs are postulated to exert an effect on target geneexpression by hybridizing with a complementary RNA sequence. The hybridRNA-ODN duplex appears to interfere with one or more aspects of RNAmetabolism including processing, translation and metabolic turnover.Chemically modified ODNs have been used to enhance nuclease stabilityand cell permeability.

Duplex DNA can be specifically recognized by oligomers based on arecognizable nucleomonomer sequence. The motif termed “GT” recognitionhas been described by Beal et al. (1992) Science, 25:1360-1363; Cooneyet al. (1988) Science, 241:456-459; and Hogan et al., EP Publication375408. In the G-T motif, the ODN is oriented antiparallel to the targetpurine-rich sequence and A-T pairs are recognized by adenine or thymineresidues and G-C pairs by guanine residues.

Sequence-specific targeting of both single-stranded and duplex targetsequences has applications in diagnosis, analysis, and therapy. Undersome circumstances wherein such binding is to be effected, it isadvantageous to stabilize the resulting duplex or triplex over long timeperiods.

Covalent crosslinking of the oligomer to the target provides oneapproach to prolong stabilization. Sequence-specific recognition ofsingle-stranded DNA accompanied by covalent crosslinking has beenreported by several groups. For example, Vlassov at al. (1986) Nuc.Acids Res., 14:4065-4076, describe covalent bonding of a single-strandedDNA fragment with alkylating derivatives of nucleomonomers complementaryto target sequences. A report of similar work by the same group is thatby Knorre et al. (1985) Biochimie, 67:785-789. It has also been shownthat sequence-specific cleavage of single-stranded DNA can be mediatedby incorporation of a modified nucleomonomer which is capable ofactivating cleavage. Iverson and Dervan (1987) J. Am. Chem. Soc.,109:1241-1243. Covalent crosslinking to a target nucleomonomer has alsobeen effected using an alkylating agent complementary to thesingle-stranded target nucleomonomer sequence. Meyer et al. (1989) J.Am. Chem. Soc., 111:8517-8519. Photoactivated crosslinking tosingle-stranded ODNs mediated by psoralen has been disclosed. Lee et al.(1988) Biochem., 27:3197-3203. Use of crosslinking in triple-helixforming probes has also been disclosed. Horne et al. (1990) J. Am. Chem.Soc., 112:2435-2437.

Use of N⁴, N⁴-ethanocytosine as an alkylating agent to crosslink tosingle-stranded and double-stranded oligomers has also been described.Webb and Matteucci (1986) J. Am. Chem. Soc., 108:2764-2765; (1986) Nuc.Acids Res., 14:7661-7674; and Shaw et al. (1991) J. Am. Chem. Soc.,113:7765-7766. These papers also describe the synthesis of ODNscontaining derivatized cytosine. The synthesis of oligomers containingN⁶, N⁶-ethanoadenine and the crosslinking properties of this residue inthe context of an ODN binding to a single-stranded DNA has beendescribed. Matteucci and Webb (1987) Tetrahedron Letters, 28:2469-2472.

In a recent paper, sequence-specific binding of an octathymidylateconjugated to a photoactivatable crosslinking agent to bothsingle-stranded and double-stranded DNA is described. Praseuth et al.(1988) Proc. Natl. Acad. Sci. (USA), 85:1349-1353. In addition,targeting duplex DNA with an alkylating agent linked through a5′-phosphate of an ODN has been described. Vlassov et al. (1988)Gene313-322; and Fedorova et al. (1988) FEBS Lett., 228:273-276.

In effecting binding to obtain a triplex, to provide for instanceswherein purine residues are concentrated on one chain of the target andthen on the. opposite chain, oligomers of inverted polarity can beprovided. By “inverted polarity” is meant that the oligomer containstandem sequences which have opposite polarity, i.e., one having polarity5′→3′ followed by another with polarity 3′→5′, or vice versa. Thisimplies that these sequences are joined by linkages which can be thoughtof as effectively a 3′—3′ internucleoside junction (however the linkageis accomplished), or effectively a 5′—5′ internucleoside junction. Sucholigomers have been suggested as by-products of reactions to obtaincyclic ODNs. Capobionco et al. (1990) Nuc. Acids Res., 18:2661-2669.Compositions of “parallel-stranded DNA” designed to form hairpinssecured with AT linkages using either a 3′—3′ inversion or a 5′—5′inversion have been synthesized. van de Sande et al. (1988) Science,241:551-557. In addition, triple helix formation using oligomerswhich.contain 3′—3′ linkages have been described. Horne and Dervan(1990) J. Am. Chem. Soc., 112:2435-2437; and Froehler et al. (1992)Biochem., 31:1603-1609.

The use of triple helix (or triplex) complexes as a means for inhibitionof the expression of target gene expression has been previously adduced(International Application No. PCT/US89/05769). Triple helix structureshave been shown to interfere with target gene expression (InternationalApplication No. PCT/US91/09321; and Young et al. (1991) Proc. Natl.Acad. Sci., 88:10023-10026), demonstrating the feasibility of thisapproach.

Various modifications have been found to be suitable for use in ODNs.Oligomers containing 5-propynyl modified pyrimidines have beendescribed. Froehler et al. (1992) Tetrahedron Letters, 33:5307-5310.2′-Deoxy-7-deazaadenosine and 2′-deoxy-7-deazaguanosine have beenincorporated into ODNs and assessed for binding to the complementary DNAsequences. Thermal denaturation analysis (Tm) has shown that thesesubstitutions modestly decrease the Tm of the duplex when these analogsare substituted for 2′-deoxyadenosine and 2′-deoxyguanosine. Seela andKehne (1987) Biochem., 26:2232-2238; and Seela and Driller (1986) Nuc.Acids Res., 14:2319-2332. It has also been shown that ODNs whichalternate 2′-deoxy-7-deaza-adenosine and -thymidine can have a slightlyenhanced duplex Tm over ODNs containing 2′-deoxy-adenosine and-thymidine. Seela and Kehne (1985) Biochem., 24:7556-7561.

2′, 3′-dideoxydeazapurine nucleosides have been used as chainterminators for DNA sequencing. 7-propargyl amino linkers are used forincorporation of fluorescent dyes into the nucleoside triphosphates

DNA synthesis via amidite and hydrogen phosphonate chemistries has beendescribed. U.S. Pat. Nos. 4,725,677; 4,415,732; 4,458,066; and4,959,463.

Prior attempts at antisense inhibition of HIV have focused on inhibitionof the synthesis of some particular viral protein thought to beessential to the success of the infection and to RNAs which are believedto have important biological function. It has now been found thatinhibition of viral gene expression and replication can be moreefficiently achieved by targeting the conserved sites of the viral RNAsthat signal the synthesis of conserved HIV proteins, particularly thep24 core antigen protein.

SUMMARY OF THE INVENTION

The present invention is directed to ODNs comprising nucleotidesequences sufficiently complementary to conserved regions of humanimmunodeficiency virus genetic material such that when bound to saidregion, the ODNs effectively prevent expression of the genetic material.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an autoradiograph of a SDS-PAGE showing in vivo synthesis ofHIV proteins and their breakdown products. FIG. 1 is described inExample 3.

FIG. 2 is an autoradiograph of a SDS-PAGE showing significant inhibitionof expression of the HIV proteins by an antisense ODN directed againstthe rev sequence. FIG. 2 is described in Example 4.

FIG. 3 is an autoradiograph of a SDS-PAGE showing significant inhibitionof expression of HIV proteins by antisense ODN directed against thefirst splice donor site of the HIV-1 genome. FIG. 3 is discussed inExample 5.

FIG. 4 is an autoradiograph of a SDS-PAGE showing the effects ofdifferent concentrations of antisense ODNS on HIV-gene productsynthesis. FIG. 4 is discussed in Example 6.

FIG. 5 is an autoradiograph of a SDS-PAGE showing the concentrationdependent inhibitory effects of ODN GPI-2A on p24 expression in HIVinfected cells.

FIG. 6 is a bar graph showing the concentration dependent inhibitoryeffects of ODN GPI-2A on p24 expression in HIV infected cells.

DETAILED DESCRIPTION OF THE INVENTION

Several conserved sites within HIV RNA have now been found to beeffective targets for the inhibition of expression of viral geneproducts by antisense ODNs and their analogues. The inhibition is basedon the capacity to block certain functions during viral replication asmeasured by production of p24. The clinical importance of p24, acleavage product of p55, is evidenced by the fact that serum levels ofantibody to p24 antigen of HIV provide evidence of the effectiveness ofimmune response to the virus as well as serving as a marker of freevirus in the serum of patients with advanced stage AIDS. Goedert et al.(1989) N. Engl. J. Med., 321:114.

According to the present invention, 20 mer/15 mer sequences weredesigned and employed as anti-HIV chemotherapeutic agents. The mechanismof action of antisense chemotherapeutics may be solely due to binding tothe MRNA or DNA so as to prevent translation or transcription,respectively. The mechanism of action may also be due to activation ofRNase H and subsequent degradation of the RNA.

The sequences are conserved in at least two different HIV isolates, and,therefore the antisense ODNs are effective agents against a wide varietyof HIV strains.

The sequences were synthesized based on the phosphoramidite chemistry ofODN synthesis on Applied Biosystems model 380D automated DNAsynthesizer. They were purified using ODN purification cartridges and/orHPLC.

In therapeutic applications, the ODNs are utilized in a mannerappropriate for treatment of a variety of conditions by inhibitingexpression of the target genetic regions. For such therapy, the ODNs,alone or in combination can be formulated for a variety of modes ofadministration, including systemic, topical or localized administration.Techniques and formulations generally can be found in Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., latestedition. The ODN active ingredient is generally combined with apharmaceutically acceptable carrier such as a diluent or excipient whichcan include fillers, extenders, binders, wetting agents, disintegrants,surface-active agents, or lubricants, depending on the nature of themode of administration and dosage forms. Typical dosage forms includetablets, powders, liquid preparations including suspensions, emulsionsand solutions, granules, capsules and suppositories, as well as liquidpreparations for injections, including liposome preparations.

For systemic administration, injection is preferred, includingintramuscular, intravenous, intraperitoneal, and subcutaneous. Forinjection, the ODNs of the invention are formulated in liquid solutions,preferably in physiologically compatible buffers such as Hank's solutionor Ringer's solution. In addition, the ODNs can be formulated in solidform and redissolved or suspended immediately prior to use. Lyophilizedforms are also included. Dosages that can be used for systemicadministration preferably range from about 0.01 mg/Kg to 50 mg/Kgadministered once or twice per day. However, different dosing schedulescan be utilized depending on (i) the potency of an individual ODN atinhibiting the activity of its target DNA or RNA, (ii) the severity orextent of the pathological disease state, or (iii) the pharmieokineticbehavior of a given ODN.

Systemic administration can also be by transmucosal or transdermalmeans, or the compounds can be administered orally. For transmucosal ortransdermal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art, and include, for example, bile salts and fusidic acidderivatives for transmucosal administration. In addition, enhancers canbe used to facilitate permeation. Transmucosal administration can bethrough use of nasal sprays, for example, or suppositories. For oraladministration, the ODNs are formulated into conventional oraladministration forms such as capsules, tablets, and tonics.

For topical administration, the ODNs of the invention are formulatedinto ointments, salves, gels, or creams, as is generally known in theart. Formulation of the invention oligomers for ocular indications isbased on standard compositions known in the art.

In addition to use in therapy, the ODNs of the invention can be used asdiagnostic reagents to detect the presence or absence of the targetnucleic acid sequences to which they specifically bind. The enhancedbinding affinity of the invention ODNs is an advantage for their use asprimers and probes. Diagnostic tests can be conducted by hybridizationthrough either double or triple helix formation which is then detectedby conventional means. For example, the ODNs can be labeled usingradioactive, fluorescent, or chromogenic labels and the presence oflabel bound to solid support detected. Alternatively, the presence of adouble or triple helix can be detected by antibodies which specificallyrecognize these forms.

The use of ODNs containing the invention substitute linkages asdiagnostic agents by triple helix formation is advantageous since triplehelices form under mild conditions and the assays can thus be carriedout without subjecting test specimens to harsh conditions. Diagnosticassays based on detection of RNA often require isolation of RNA fromsamples or organisms grown in the laboratory, which is laborious andtime consuming, as RNA is extremely sensitive to ubiquitous nucleases.

The ODN probes can also incorporate additional modifications such asmodified sugars and/or substitute linkages that render the ODNespecially nuclease stable, and would thus be useful for assaysconducted in the presence of cell or tissue extracts which normallycontain nuclease activity. ODNs containing terminal modifications oftenretain their capacity to bind to complementary sequences without loss ofspecificity. Uhlmann et al. (1990) Chem. Rev., 90:543-584. As set forthabove, the invention probes can also contain linkers that permitspecific binding to alternate DNA strands by incorporating a linker thatpermits such binding. Froehler et al. (1992) Biochem., 31:1603-1609; andHorne et al. (1990) J. Am. Chem. Soc., 112:2435-2437.

Incorporation of base analogs into probes that also contain covalentcrosslinking agents has the potential to increase sensitivity and reducebackground in diagnostic or detection assays. In addition, the use ofcrosslinking agents will permit novel assay modifications such as (1)the use of the crosslink to increase probe discrimination, (2)incorporation of a denaturing wash step to reduce background and (3)carrying out hybridization and crosslinking at or near the meltingtemperature of the hybrid to reduce secondary structure in the targetand to increase probe specificity. Modifications of hybridizationconditions have been previously described. Gamper et al. (1986) Nuc.Acids Res., 14:9943.

ODNs of the invention are suitable for use in diagnostic assays thatemploy methods wherein either the oligomer or nucleic acid to bedetected are covalently attached to a solid support as described in U.S.Pat. No. 4,775,619. The ODNs are also suitable for use in diagnosticassays that rely on polymerase chain reaction (PCR) techniques toamplify target sequences according to methods described, for instance,in European Patent Publication No. 0 393 744. ODNs of the inventioncontaining a 3′ terminus that can serve as a primer are compatible withpolymerases used in PCR methods such as the Taq or Vent™ (New EnglandBiolabs) polymerase. ODNs of the invention can thus be utilized asprimers in PCR protocols.

The ODNs are useful as primers that are discrete sequences or as primerswith a random sequence. Random sequence primers can be generally about6, 7, or 8 nucleomonomers in length. Such primers can be used in variousnucleic acid amplification protocols (PCR, ligase chain reaction, etc.)or in cloning protocols. The substitute linkages of the inventiongenerally do not interfere with the capacity of the ODN to function as aprimer. ODNs of the invention having 2′-modifications at sites otherthan the 3′ terminal residue, other modifications that render the ODNRNase H incompetent or otherwise nuclease stable can be advantageouslyused as probes or primers for RNA or DNA sequences in cellular extractsor other solutions that contain nucleases. Thus, the ODNs can be used inprotocols for amplifying nucleic acid in a sample by mixing the ODN witha sample containing target nucleic acid, followed by hybridization. ofthe ODN with the target nucleic acid and amplifying the target nucleicacid by PCR, LCR or other suitable methods.

The ODNs derivatized to chelating agents such as EDTA, DTPA or analogsof 1,2-diaminocyclohexane acetic acid can be utilized in various invitro diagnostic assays as described in, for instance, U.S. Pat. Nos.4,772,548, 4,707,440 and 4,707,352. Alternatively, ODNs of the inventioncan be derivatized with crosslinking agents such as5-(3-iodoacetamidoprop-1-yl)-2′-deoxyuridine or5-(3-(4-bromobutyramido)prop-1-yl)-2′-deoxyuridine and used in variousassay methods or kits as described in, for instance, InternationalPublication No. WO 90/14353.

In addition to the foregoing uses, the ability of the oligomers toinhibit gene expression can be verified in in vitro systems by measuringthe levels of expression in subject cells or in recombinant systems, byany suitable method. Graessmann et al. (1991) Nuc. Acids Res., 19:53-59.In the present case, levels of p24 have been measured as indicative ofvirus replication.

All references cited herein are incorporated herein by reference intheir entirety.

The first embodiment of the present invention is an ODN complementary tothe region between the 5′ long terminal repeat (LTR) and the firstinitiation codon (AUG) of the a gene. This region contains highlyconserved sequences required for efficient viral RNA packaging. Klotmanand Wong-Staal (1991) in: The Human Retroviruses by Gallo & Jay, eds.Acad. Press. The antisense ODN is referred to as “anti-gag.” The ODN isof sufficient length and complementarity to inhibit expression of thegag gene. The complementary site is from bases +262 to +281 as numberedaccording to Ratner et al. (1985) Nature, 313:277-283. In a preferredembodiment the anti-gag ODN has the specific sequence:

5′ CCGCCCCTCGCCTCTTGCCG 3′ SEQ ID NO:1

The second embodiment of the present invention is an ODN complementaryto the sequence immediately downstream of the major splice acceptor sitebut upstream of the AUG initiation codon of the tat gene (3′ ofnucleotide 5358). Translation of this transcript is essential forefficient viral gene expression and replication. The antisense ODN isreferred to as “anti-gag-pol.” The ODN is of sufficient length andcomplementarity to inhibit expression of the gag-pol gene. Thecomplementary site is from bases +5399 to +5418 as numbered according toRatner et al. (1985) Nature, 13:277-283. In a preferred embodiment theanti-gag-pol ODN has the sequence:

5′ GGCTCCATTTCTTGCTCTCC 3′ SEQ ID NO:2

The third embodiment of the present invention is an ODN complementary tothe rev gene which is involved in the regulated expression of HIVstructural genes. Feinberg et al. (1986) Cell, 4:807; and Sodroski etal. (1986) Nature, 321:412. It has previously been observed thatcytoplasmic RNAs that encode the virion structural proteins gag, pol andenv are not found in the absence of a functional rev gene product.Sodroski et al. (1986) Knight at al. (1987) Science, 236:837-840; Malimet al. (1988) Nature, 335:181-183; and Hadzopoulou-Cladaras et al.(1989) J. Virol., 63:1265-1274. rev mutants of HIV-1 are incapable ofinducing the synthesis of the viral structural proteins and aretherefore replication defective. Sadaie et al. (1988) Science, 239:910.rev is, therefore, said to be important in governing the transition fromthe expression of the early regulatory genes to that of the latestructural genes. Greene (1991) in Mechanisms of Disease. Ed. by F.Epstein. The antisense ODN is referred to as “anti-rev.” The ODN is ofsufficient length and complementarity to inhibit expression of the revgene. The complementary site is from bases +5552 to +5566 as numberedaccording to Ratner et al. (1985) In a preferred embodiment the anti-revODN has the following sequence:

5′ CCGCTTCTTCCTGCC 3′ SEQ ID NO:3

The fourth embodiment of the present invention is an ODN complementaryto the region within the second splice acceptor site. This regioncontains highly conserved sequences required for efficient viral RNApackaging. Klotman and Wong-Staal (1991) in: The Human Retroviruses byGallo & Jay, eds. Acad. Press. The antisense ODN is referred to as“GPI-2A.” The ODN is of sufficient length and complementarity to inhibitexpression of the gag gene. The complementary site is from bases +1189to +1208 as numbered according to Ratner et al. (1985). In a preferredembodiment the anti-gag ODN has the specific sequence:

5′ CCGCCCCTCGCCTCTTGCCG 3′ SEQ ID NO:1

In a further preferred embodiment, the ODNS were chemically modified bysubstitution of the naturally occurring oxygen of the phosphodiesterbackbone with sulfur to form the corresponding phosphorothioatederivatives of the oligomers. The positions of the sulfur are as shownbelow.

Anti-gag:

5′ C^(S)CG^(S)CC^(S)CC^(S)TC^(S)GC^(S)CTC^(S)TTG^(S)CC^(S)G 3′ SEQ IDNO:4;

Anti-gagpol:

5′ G^(S)GC^(S)TC^(S)CA^(S)TTTC^(S)TTG^(S)CTC^(S)TC^(S)C 3′ SEQ ID NO:5;

Anti-rev:

5′ C^(S)CG^(S)C^(S)TTCTTC^(S)C^(S)TGC^(S)C 3′SEQ ID NO:6; and GPI-2A:

5′ G^(S)GTTC^(S)TTTTG^(S)GTCC^(S)TTG^(S)TC^(S)T 3′ SEQ ID NO:7.

In accordance with the present invention, methods of modulating theexpression of the p24 protein are provided. The targeted RNA, or cellscontaining it, are treated with the ODN analogs which bind to specificregions of the RNA coding for the HIV p24 core structural protein. TheRNA targeted sites include regions involved in the mechanism ofexpression of the HIV p24 core structural protein.

The following examples are intended to illustrate, but not to limit, theinvention. Efforts have been made to insure accuracy with respect tonumbers used (e.g., amounts, temperatures, etc.), but some experimentalerrors and deviations should be taken into account. Unless indicatedotherwise, parts are parts by weight, temperature is in degreesCentigrade, and pressure is at or near atmospheric.

EXAMPLE 1 Cell Culture

To determine the effect of antisense oligomer on viral gene expression,B4.14 cells, provided by Dr. David Rekosh, Microbiology Department,University of Virginia, were seeded at a cell density of 5,000-12,000cells per well in 24-well/35 mm plastic tissue culture plates and weremaintained in Iscove's Modified Dulbecco's Medium with 10% calf serum,50 μg/ml gentamycin and 200 μg/ml hygromycin B at 37° C. in a humidifiedincubator with 5% CO₂ for a few hours. Subsequently, the incubated cellswere washed and incubated under the same conditions with mediumcontaining the indicated concentrations of ODN and 10% serum heatinactivated to reduce serum nuclease activity. The same ODN sequences,but with switched polarity were used as controls.

EXAMPLE 2 Viral Antigen Assay

Cells cultured as described in Example 1 were labeled with 75 to 150μCi/ml [³⁵S]-methionine (70% L-Methionine/15% L-Cysteine) in thepresence of methionine-free medium containing 29.2 mg/100 ml glutamine,50 μg/ml gentamycin, 200 μg/ml hygromycin B. 10% heat inactivated fetalcalf serum plus the desired concentration of oligomer. The[³⁵S]-methionine concentration was 185 MBq and the specific activity was1057 Ci/mmole). Labeled samples were subsequently washed with phosphatebuffered saline (PBS) and resuspended in 200 μl lysis buffer comprisedof 50 mM Tris, pH 7.2; 150 mM NaCl; 5 mM EDTA; 1% Triton-100; 0.2%Deoxycholic acid.

Culture medium containing labeled virus was treated with 10% TritonX-100 to a 1% final concentration to disrupt virus particles. Thesamples were preabsorbed with protein A-Sepharose beads for 30 min. at4° C. [³⁵S ]-methionine-labeled viral proteins were thenimmunoprecipitated for 2 hours using protein A-Sepharose beads and 2.5μl/sample of polyclonal rabbit antiserum directed against HIV-1 p25/24,obtained from the National Institute of Allergy and Infectious Diseases(AIDS Research and Reference Reagent Program). The antibodies wereobtained from National Institute of Allergy & Infectious Disease (AIDSResearch & Reference Reagent Program) and MicroGeneSys, Inc.

The resulting pellets were washed 4 times with lysis buffer, once withlysis buffer containing 500 mM NaCl and finally once with TNE buffercomprised of 10 mM Tris, pH 7.2; 25 mM NaCl; 1 mM EDTA. Samples werethen resuspended in 20-30 μl 2X SDS sample buffer, boiled for 5-10 min,applied to a 12.5% SDS polyacrylamide gel electrophoresis and thenanalyzed by electrophoresis, according to the method described byLaemmli (1970) Nature, 227:680-685. The results obtained are listed inTable 1 and in FIGS. 1-3. Percent inhibition is determined bydensitometric analysis of the autoradiography. The first two ODNs(anti-gag and anti-tat) were phosphorothioate derivatives [sulfurizationon alternate bases]. Inhibition was observed at ODN concentrations of 5μM assayed after 3 days incubation with the oligomer. The third oligomerwas a 15-mer phosphodiester derivative. Observed inhibition was atoligomer concentration of 200 μg/ml assayed after 6 days incubation withthe oligomer.

TABLE 1 Preliminary Observation Inhibition of viral protein synthesis byantisense oligomer in B4.14 cells Complementary % in- Sequence 5′-3′Site Function hibition CCGCCCCTCGCCTCTTGCCG 262-281 Splice 30 DonorGGCTCCATTTCTTGCTCTCC 5399-5418 tat 30 initiator CCGCTTCTTCCTGCC5552-5566 Rev 40

EXAMPLE 3

FIG. 1 is an autoradiograph of a SDS-PAGE showing in vivo synthesis ofHIV-1 viral proteins and their breakdown products. The following sampleswere run. Two hundred 1μof CMT3 [wild-type (left)] and B4.14[transfected line (right)] cell lysates following metabolic labelingwith [³⁵S]-methionine were immunoprecipitated with rabbit serum againstp24 viral antigen as described above. The positions of the viralproteins (p160; p55 and p24) are clearly visible in the B4.14 celllysate but not in control cell line CMT3 cell lysate.

EXAMPLE 4

FIG. 2 is an autoradiograph of a SDS-PAGE showing a significantinhibition of expression of HIV proteins by the antisense ODN directedto the rev sequence. The following experiment was performed. Two hundredμl of B4.14 [transfected line] cell lysates following 3 days treatmentwith antisense [AS]; and sense, the inverse complement of the antisenseoligomer [CS]; and subsequent [³³S]-methionine labeling wereimmunoprecipitated with rabbit serum directed against p24 viral antigenas described above. Equal amounts of protein were loaded on each lane.

EXAMPLE 5

FIG. 3 is an autoradiograph of a SDS-PAGE showing a significantinhibition of expression of HIV proteins by the antisense ODN directedto the first splice site donor of the HIV-1 genome. The followingexperiment was performed. Two hundred μl of B4.14 [transfected cellline] cell lysates/medium following 6 days treatment with antisense[AS]; sense, the inverse complement of the antisense ODN [S]; andcontrol [B4.14] cell lysate only); and subsequent ³⁵S-methioninelabeling were immunoprecipitated with rabbit serum directed against p24viral antigen as described above. Equal amounts of protein were added ineach lane.

EXAMPLE 6 The Effects of Different

Concentrations of the Antisense ODNs

To determine whether there was a dose relationship of the antisense ODNson HIV gene expression, the following experiment was performed.

The cells were cultured as described in Example 1 and incubatedovernight with different concentrations of ODNs directed against thefirst splice donor site in the presence of 5 μg/ml Lipofectin and 1%heat-inactivated fetal calf serum. The medium was subsequently replacedwith fresh medium containing 10% heat-inactivated serum. ODN was thenadded and incubated for 7 days. Western blot analysis was performed withrabbit polyclonal antibody directed against HIV p24/55 proteins.

Following SDS-polyacrylamide electrophoresis, cellular proteins wereelectrophoretically transferred to Immobilon membrane (Schleicher andSchuell) as follows. An Immobilon membrane was placed in methanol in aclean dish, washed several times in deionized distilled water and soakedin western transfer buffer (60.6 g Tris-HCl; 288 g glycine; 4 l methanoland distilled water to 20 l ). The apparatus used is the Dio-RadTrans-Blot cell, used according to the manufacturer's instructions.Western blot analysis was performed using the Vectastain ABC kit(Alkaline Phosphatase Rapid IgG) (Vector Laboratories) according to themanufacturer's instructions.

The results are shown in FIG. 4 where it can be seen that 0.5 and 1 μMantisense ODN are equally effective at preventing p24 synthesis.

EXAMPLE 7 The Effects of Different Concentrations of the ODN GPI-2A

To determine the ability of the ODN GPI-1A to inhibit expression of p24in HIV infected cells, the following experiments were performed.

Cells were incubated overnight with 0.1, 0.5 and 1.0 μM of the ODN inthe presence of 1% heat-inactivated fetal calf serum. The serumconcentration was subsequently raised to 10% and incubated for 3 days.About 3×10⁷ cpm/probe was immunoprecipitated using rabbit polyclonalantibody directed against p24/25 viral proteins as described above. Thelane marked control had the sense strand, the inverse complement of theantisense oligomer, added to the cells rather than the sense strand. Theautoradiograph in FIG. 5 shows that there was a dose-dependentinhibition of the HIV viral core antigen, among others.

The autoradiograph was then subjected to densitometry analysis. Theresults, presented in FIG. 6, indicate that at 1.0 μM, the ODN inhibitedabout 50% of the p24synthesis.

7 1 20 DNA Artificial Sequence Synthetic Construct 1 ccgcccctcgcctcttgccg 20 2 20 DNA Artificial Sequence Synthetic Construct 2ggctccattt cttgctctcc 20 3 15 DNA Artificial Sequence SyntheticConstruct 3 ccgcttcttc ctgcc 15 4 20 DNA Artificial Sequence SyntheticConstruct 4 ccgcccctcg cctcttgccg 20 5 20 DNA Artificial SequenceSynthetic Construct 5 ggctccattt cttgctctcc 20 6 15 DNA ArtificialSequence Synthetic Construct 6 ccgcttcttc ctgcc 15 7 20 DNA ArtificialSequence Synthetic Construct 7 ggttcttttg gtccttgtct 20

What is claimed is:
 1. At least one oligonucleotide comprising a nucleotide sequence sufficiently complementary to a region of human immno-deficiency virus genetic material such that when bound to said region, the oligonuclcotide inhibits expression of the genetic material wherein the nucleotide sequence is complementary to the nucleic acid sequences selected from the group consisting of: +5399 to +5418, and +5552 to +5566.
 2. The at least one oligonucleotide according to claim 1 wherein the nucleotide sequence is selected from the group consisting of: 5′ GGCTCCATTTCTTGCTCTCC 3′ (SEQ ID NO:2); and 5′ CCGCTTCTTCCTGCC 3′ (SEQ ID NO:3).
 3. The at least one oligonucleotide according to claim 1 wherein the nucleotide sequence is selected from the group consisting of; 5^(S) G^(S)GC^(S)TC^(S)CA^(S)TTTC^(S)CTC^(S)TC^(S)C 3′ (SEQ ID NO:5); and 5^(S)C^(S)CG^(S)C^(S)TTCTTC^(S)C^(S)TGC^(S)C 3′ (SEQ ID NO:6) wherein S stands for a sulfur atom.
 4. A composition comprising an oligonucleotide and according to claim 1 or 2 a pharmaceutically acceptable carrier therefor.
 5. The composition according to claim 4, further comprising a liposome preparation.
 6. The composition according to claim 5, wherein the liposome preparation comprises lipofectin. 